Wounds can be divided into two major categories: acute wounds, such as those associated with surgical incisions, and chronic wounds, such as those associated with diabetes.
Acute wound healing has been categorized into four phases: coagulation, inflammation, proliferation, and remodeling. At the time of injury, coagulation is initiated by activated platelets binding thrombin and forming a plug. Vasoconstriction and cytokine release (e.g., platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-β)) also occur. During the second phase, inflammatory cells, such as macrophages and polymorphonuclear (PMN) cells are recruited, which phagocytose bacteria and produce additional cytokines and growth factors such as IL-6 and TGF-β. Fibroblasts are also recruited and produce extracellular matrix components such as fibronectin and collagen. Matrix metalloproteinases (MMPs) are produced by inflammatory cells, and help prepare the wound for angiogenesis (new blood vessel formation). In the proliferation phase, fibroblasts and endothelial cells proliferate, and fibroblasts secrete extracellular matrix proteins forming granulation tissue. Later, fibroblasts remodel tissue. The granulation tissue serves as a matrix over which the keratinocytes migrate to create a new surface across the wound, i.e., a new epidermis. The wound contracts and later a scar is formed. During the final remodeling phase, collagen fibrils in the scar are degraded by MMPs.
Unlike acute wound healing, chronic wound healing does not proceed normally through the four healing phases. Defects in the healing process result in inflammatory excess (e.g., excessive production of Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and MMPs), a deficiency of important growth factors needed for proper healing, bacterial overgrowth and senescence of fibroblasts. Further, an epithelial layer fails to cover the entire surface of the wound and, consequently, a chronic wound remains open and subject to infection. Bacteria colonize the chronic wound beneath a biofilm layer (which they secrete), activate virulence factors, and trigger NFκB-dependent inflammatory pathways, thereby continuing the process of inflammatory excess that prevents proper healing of the wound.
Chronic diabetic wounds (ulcers) are a common and serious complication of diabetes. Failure to heal wounds from overwhelming injury or impaired healing, as in diabetic wounds, has become an increasingly serious global problem with significant morbidity and expense. Clark, R. A. et al. (2007) J. Invest. Dermatol. 127:1018-1029. There are nearly 20 million diagnosed type II diabetics in the US, and up to 15% of these patients will develop a chronic wound. Of these, almost 20% will require amputation for this problem. Albert S, Clin Podiatr Med. Surg. 2002, 19:483-491. These complications not only cause pain and loss of function, but also represent a significant financial burden to the health care industry, with an annual expenditure of 1.45 billion dollars in the Medicare system alone. Reiber G E L B, Gibbons G W, Am J. Surg. 1998, 176:5 S-10S.
Nearly three quarters of diabetic individuals have peripheral neuropathy, trauma (usually repetitive trauma of daily ambulation), ulcerations (particularly of the plantar surface of the foot), and faulty healing. Peripheral neuropathy leads to a loss of sensation and renders diabetic patients more susceptible to recurrent mechanical injury. For example, a diabetic person does not receive feedback sensation from an ill-fitting shoe that causes a pressure point. Accordingly, the pressure point is not-relieved and can progress to a partial or full thickness skin injury. Due to faulty wound healing, the injury becomes a chronic wound in a diabetic patient that can involve subcutaneous tissue, or even bone. This can lead to osteomyelitis, gangrene and necrosis, and amputation.
Histologically, diabetic wounds exhibit impairments in the remodeling of the dermis and are hypocellular, hypovascular, and show impaired ability to form granulation tissue. Because of these underlying problems, the wounds have difficulty inducing re-epithelialization and experience delayed closure, likely due to a defective matrix, which would normally facilitate migration of the cellular elements. Many of these problems are related to the effects that diabetes has on cellular functioning. Diabetic fibroblasts show impaired ability to migrate, proliferate, and produce less growth factors and VEGF (critical to neovascularization and angiogenesis) than normal fibroblasts. Lerman et al., Am J Pathol 2003, 162:303-312. This suggests a mechanism for the histological lack of granulation tissue formation, as there would be a paucity of cells migrating into the wound and nutrients brought by the blood supply to aid in healing the wound. Macrophages, which are essential to the formation of granulation tissue and debris clearance, also show impaired migration into diabetic wounds. Wetzler et al., Large and Sustained Induction of Chemokines during Impaired Wound Healing in the Genetically Diabetic Mouse Prolonged Persistence of Neutrophils and Macrophages during the Late Phase of Repair, 2000, 115:245-253. There is also a chronic presence of MMPs that continue to degrade newly formed granulation tissue.
Furthermore, diabetes results in a state of impaired angiogenesis as well as chronic vascular inflammation. The altered glucose and free fatty acid (FFA) metabolism of diabetes results in oxidative stress, endothelial dysfunction and activation of inflammatory cytokines, particularly those regulated by NFκB. Increased expression of TNFα, IL-6 and other inflammatory factors has been demonstrated, and the increased expression leads to a hyperinflammatory state that prevents closing of the wound.
Although novel wound healing agents, skin substitutes, and devices are continually emerging, their utility for clinical application remains limited, as few chronic wounds are actually healed. Existing pharmaceutical agents, such as Regranex® gel, are currently used to treat acute and chronic wounds. Regranex® gel contains becaplermin, a recombinant human platelet-derived growth factor isoform dimer, BB (PDGF-BB), which promotes cellular proliferation of the cells of the dermis, which are mainly fibroblasts, and angiogenesis. It is indicated for the treatment of lower extremity diabetic neuropathic ulcers that extend into the subcutaneous tissue or beyond and have an inadequate blood supply. An increased rate of malignancies and death in patients using Regranex® gel has been reported, indicating that safer alternatives to this drug are needed. Other proteins that have shown promise in vivo include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor-β (TGF-β) and others. Galiano et al., Am J Pathol 2004, 164:1935-1947; Michaels et al., Wound Repair and Regeneration 2005, 13:506-512; Obara et al., Wound Repair and Regeneration 2005, 13:390-397; Greenhalgh et al., Am J Pathol 1990, 136:1235-1246; Acosta et al., International Wound Journal 2006, 3:232-239.
A role for calreticulin, a 46 kDa protein (it resolves at a higher molecular weight in SDS-page, e.g., 55-60 kDa) associated with hyaluronan, in the treatment of acute wounds (reduced scar formation), such as surgical wounds and wounds incurred in accidental trauma, has been described by the present inventors. See, e.g., U.S. Pat. No. 5,591,716. Calreticulin is a highly conserved major calcium-binding protein of the endoplasmic reticulum (ER). The heralded functions of calreticulin are intracellular, in calcium homeostasis and in binding N-linked oligosaccharide protein intermediates to ensure proper glycoprotein conformation in the ER. There is 90% amino acid homology conservation across species. Johnson, S. et al. (2001) Trends Cell Biol. 11:122-129; Bedard, K. et al. (2005) Int. Rev. Cytol. 11:122-129; Sezestakowska, D. et al. (2006) International Workshop on Calreticulin, Niagara Falls, Canada. 1:135-139; Gold, L. I. et al. (2006) J. Investig. Dermatol. Symp. Proc. 11:57-65.
Chronic wounds and their management are very different than acute wounds and, thus, therapeutic agents that are useful for the treatment of acute wounds may not be as useful for the treatment of chronic wounds. Thus, there remains a need to discover new therapeutic agents and methods of treatment that are useful for the healing of chronic wounds, including chronic diabetic wounds.